WO2012139591A1 - Surveillance de la fibrose hépatique chez un patient infecté par le virus de l'hépatite c - Google Patents

Surveillance de la fibrose hépatique chez un patient infecté par le virus de l'hépatite c Download PDF

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Publication number
WO2012139591A1
WO2012139591A1 PCT/DK2012/050127 DK2012050127W WO2012139591A1 WO 2012139591 A1 WO2012139591 A1 WO 2012139591A1 DK 2012050127 W DK2012050127 W DK 2012050127W WO 2012139591 A1 WO2012139591 A1 WO 2012139591A1
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WIPO (PCT)
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level
sample
fibrosis
liver
liver fibrosis
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PCT/DK2012/050127
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English (en)
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Morten Ruhwald
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Hvidovre Hospital
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Publication of WO2012139591A1 publication Critical patent/WO2012139591A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/521—Chemokines
    • G01N2333/522—Alpha-chemokines, e.g. NAP-2, ENA-78, GRO-alpha/MGSA/NAP-3, GRO-beta/MIP-2alpha, GRO-gamma/MIP-2beta, IP-10, GCP-2, MIG, PBSF, PF-4 or KC

Definitions

  • the present invention relates to a method for monitoring the degree of liver fibrosis in a hepatitis C infected patient by spotting a blood sample obtained from a human infected with hepatitis C virus on a filter paper for subsequent analysis.
  • HCV infection is the number one cause of liver cancer and liver transplantation. HCV infection is often asymptomatic, but once established, chronic infection can progress to scarring of the liver (fibrosis & cirrhosis), liver failure and liver cancer.
  • Chronic HCV infection can be treated e.g. with pegylated-interferon and ribavirin and patients who develop advanced fibrosis are offered treatment.
  • the treatment takes a long time (24-48 weeks), is expensive and has severe side effects.
  • IP- 10 probably is the best stand-alone biomarker for
  • WO08028489 made by the present inventors relates to a method for measuring cell-mediated immune reactivity (CMI) in mammals based on the production of IP- 10, but does not relate to direct measurement of the spontaneous IP-10 concentration and correlation with HCV without prior antigen stimulation of the sample.
  • CMI cell-mediated immune reactivity
  • WO09060317 relates to a method of collecting and analyzing a bodily fluid from a subject's nasal cavity by use of a filter paper, and this sampling of nasal fluids is then used for subsequent assessment of biomarkers for the diagnosis and monitoring.
  • IP-10 IP-10
  • HCV HCV
  • Mei et al disclose that in general, any analyte that can be measured from whole blood, serum or plasma potentially also could from a generic or theoretical point of view be measured from dried blood on filter paper. However, the article does not specifically disclose any content relating to HCV or IP-10.
  • WO 2008/064684 relates to a method comprising mixing blood or another biological fluid sample with a test compound and spotting the blood or the biological fluid on filter paper for subsequent analysis of the effect of the test compound on the blood or on the biological fluid.
  • this application relates to cell mediated immune response and the appliance of desired sample preparation by storing a blood sample on a filter paper and the subsequent analysis.
  • the disclosure does not disclose any content relating to HCV or IP-10.
  • cytokines are labile and short lived, resulting in degradation during pre-analytical handling, incubation, storage and transport. For this reason, comparative analyses and diagnostic tests have to be carried out immediately upon blood collection and incubation in central laboratories. This is not always practical, e.g. when taking blood samples in remote areas, when doing in vitro and in vivo time-studies or when comparing samples from many different individuals.
  • One solution to this problem is to freeze samples for transport and storage. This, however, does not guarantee preservation of constituents, requires large freezing, transport and storage capacity, requires thawing each time an analysis is performed, and is vulnerable with regard to shortage of electric power supply.
  • IL-6 is a well-known cytokine and biomarker for the purpose of measurement of infection and is applicable on a blood/serum/plasma level, but not applicable for diagnosis on a dried blood spot level (cf. Skogstrand et al. Clinical Chemistry 2005)
  • PPD purified protein derivative
  • BCG Mycobacterium bovis bacille-Calmette Guerin
  • LPS bacterial lipopolysaccharide
  • IFN- ⁇ is a biomarker know to be very stable in plasma with prolonged storage e.g. at -20°C for 2 years (De Jager W, BMC immunology 2009, 10: 52) or up to 2 weeks at +2 to +8°C (published on the website of Cellestis). Nevertheless, Skogstrand et al (J.immunol. Methods 2008, table 4) demonstrate that IFN- ⁇ levels in dried plasma spots as unstable and unpredictable. Stored at room temperature, the IFN- ⁇ levels increase 1.4 fold after 24 hours and double after 30 days storage. Stored at 4°C for 7 days, IFN- ⁇ levels decrease to 60% of the initial levels.
  • IL-12 is known to be very stable in plasma with prolonged storage (De Jager W, BMC immunology 2009, 10: 52). Despite the robust nature of IL-12, Skogstrand et al (J.immunol. Methods 2008) demonstrate a 1.2 fold increase at 24 hours storage at room temperature, increasing to 1.9 fold after 7 days and 2.6 fold after 30 days. The increase in IL-12 is even more dramatic at 37°C where Skogstrand et al (J.immunol. Methods 2008) demonstrate a 7.4 fold increase after 30 days storage.
  • an object of the present invention relates to monitoring liver fibrosis using IP-10.
  • one aspect of the invention relates to a method for monitoring the degree of liver fibrosis in a hepatitis C infected patient by spotting a blood sample obtained from a human infected with hepatitis C virus on a filter paper for subsequent analysis of the correlation between the IP-10 level and the degree of liver fibrosis.
  • Figure 1 compares two groups of patients with HCV; one with little/no fibrosis and one with advanced liver fibrosis. As can be clearly appreciated there is a difference between the two groups of patients.
  • Figure 2 compares the two groups of patients with HCV; one with little/no fibrosis and one with advanced liver fibrosis. As can be clearly appreciated there is a difference between the two groups of patients.
  • Figure 4 is a receiver operation characteristic curve analysis comparing the ability of the plasma and the filter paper method of IP-10 determination to differentiate between little/no fibrosis (specificity) and cirrhosis (sensitivity). As can be appreciated from the figure both methods are able to differentiate between the two groups of patients, if further appears that the filter paper method is superior to the plasma method, although this difference failed to reach significance in this material (Hanley-McNeal's test).
  • Hepatitis C is an infectious disease affecting the liver, caused by the hepatitis C virus (HCV).
  • HCV hepatitis C virus
  • the infection is often asymptomatic, but once established, chronic infection can progress to scarring of the liver (fibrosis), and advanced scarring (cirrhosis), which is generally apparent after many years. In some cases, those with cirrhosis will go on to develop liver failure or other complications of cirrhosis, including liver cancer or life threatening esophageal varices and gastric varices.
  • the hepatitis C virus is spread by blood-to-blood or sexual contact. Most people have few, if any symptoms after the initial infection, yet the virus persists in the liver in about 85% of those infected.
  • HCV Antigen assays can be used for screening of high-risk patients such as dialysis patients, IV drug users, paediatric samples and organ donors among others.
  • clinical utility of a HCV antigen assay is primarily to significantly reduce the diagnostic window compared to Antibody only HCV assays by detecting an active infection. It provides an alternative to the traditionally expensive and slower NAT testing and opens new horizons in the diagnosis of infection.
  • Chronic hepatitis C is typically defined as infection with the hepatitis C virus persisting for more than six months, but there might exist variations between various physicians.
  • Chronic hepatitis C is an infection that persisted for more than 6 months. Clinically, it is often asymptomatic, and it is mostly discovered accidentally (e.g. at a routine check-up).
  • the present invention enables the skilled addressee with the option of monitoring all clinical degrees of chronic hepatitis C.
  • Factors that have been reported to influence the rate of HCV disease progression include age (increasing age associated with more rapid progression), gender (males have more rapid disease progression than females), alcohol consumption (associated with an increased rate of disease progression), HIV co-infection (associated with a markedly increased rate of disease progression), and fatty liver (the presence of fat in liver cells has been associated with an increased rate of disease progression).
  • hepatitis C is a systemic disease and patients may experience a wide spectrum of clinical manifestations ranging from an absence of symptoms to a more symptomatic illness prior to the development of advanced liver disease.
  • Generalized signs and symptoms associated with chronic hepatitis C include fatigue, flu-like symptoms, joint pains, itching, sleep disturbances, appetite changes, nausea, and depression.
  • signs and symptoms may appear that are generally caused by either decreased liver function or increased pressure in the liver circulation, a condition known as portal hypertension.
  • liver cirrhosis Possible signs and symptoms of liver cirrhosis include ascites (accumulation of fluid in the abdomen), bruising and bleeding tendency, varices (enlarged veins, especially in the stomach and esophagus), jaundice, and a syndrome of cognitive impairment known as hepatic encephalopathy. Hepatic encephalopathy is due to the accumulation of ammonia and other substances normally cleared by a healthy liver. Liver enzyme tests show variable elevation of ALT and AST. Periodically, they might show normal results. Usually prothrombin and albumin results are normal, but may become abnormal, once cirrhosis has developed. The levels of elevation of liver tests do not correlate well with the amount of liver injury on biopsy. Viral genotype and viral load also do not correlate with the amount of liver injury.
  • Liver biopsy is the best test to determine the amount of scarring and inflammation. Radiographic studies, such as ultrasound or CT scan, do not always show liver injury until it is fairly advanced. However, non-invasive tests typically based on a blood sample are emerging, with IP-10, and FibroTest one can now estimate liver fibrosis.
  • Chronic HCV infection can potentially be treated with anti-viral medication e.g. pegylated interferon-alpha-2a or pegylated interferon-alpha-2b (brand names Pegasys or PEG-Intron) and the antiviral drug ribavirin for a period of 24 or 48 weeks, depending on hepatitis C virus genotype.
  • anti-viral medication e.g. pegylated interferon-alpha-2a or pegylated interferon-alpha-2b (brand names Pegasys or PEG-Intron) and the antiviral drug ribavirin for a period of 24 or 48 weeks, depending on hepatitis C virus genotype.
  • Boceprevir, Sitagliptin and new types of inferferon drugs are emerging as new treatment modalities. All treatments have severe side effects.
  • GT genotype
  • Sustained cure rates (sustained viral response) of 75% or better are seen in people with HCV genotypes 2 and 3 with 24 weeks of treatment. Patients achieving HCV RNA below 1000 IU/mL by day 7 (i.e. just prior to the second dose of pegylated interferon) may be treated for as little as 12 weeks with retained sustained cure rates. Sustained response is about 50% in patients with HCV genotype 1 given 48 weeks of treatment. In patients with HCV genotype 1, if treatment with pegylated interferon + ribavirin does not produce a 2-log viral load reduction or complete clearance of RNA (termed "early virological response") after 12 weeks the chance of treatment success is less than 1%.
  • Sustained response is about 65% in those with genotype 4 given 48 weeks of treatment.
  • genotype 6 disease The evidence for treatment in genotype 6 disease is currently sparse, and the evidence that exists is for 48 weeks of treatment at the same doses as are used for genotype 1 disease. Physicians considering shorter durations of treatment (e.g., 24 weeks) should do so within the context of a clinical trial.
  • the present invention is the first to demonstrate that the correlation between IP- 10 and fibrosis development can be monitored on a blood spotted filter paper and that IP-10 remains significantly detectable, thus in one embodiment, the present invention relates to a method for monitoring the degree of liver fibrosis in a hepatitis C infected patient comprising the steps of a) spotting a blood sample obtained from a human infected with hepatitis C virus on a filter paper for subsequent analysis b) determining the IP-10 level in said spotted blood sample c) comparing the determined IP-10 level with a reference- 1 eve I d) determining the degree of liver fibrosis by correlating the determined IP- 10 level with the reference level and indicating whether said patient has
  • the method may be used to determine the progression of the liver fibrosis in a hepatitis C infected patient by comparing the levels of liver fibrosis in samples obtained from the patient at different time point.
  • the method is used to determine the extent of liver fibrosis is a hepatitis C infected patient with reference to a standard sampel or predetermined standard level.
  • IP-10 is very stable in dried plasma spots on filter paper.
  • the stability data combined with the clinical evidence underpins the strength of the method (cf. Example 6).
  • Hepatic fibrosis is a process of scarring through fibrous tissue deposit which results in the destruction of the parenchyma, with the ultimate progressive stage being cirrhosis.
  • Liver fibrosis is traditionally monitored liver biopsy. Liver biopsy is an imperfect tool; due to sampling errors, biopsy size (5 to 30 mm) and intra- and inter- observer variability, it is now agreed that biopsy is an "imperfect Gold Standard". Biopsy continues to present inconveniences: 30% of patients complain of pain, up to 3% have been noted to have complications severe enough to require
  • liver biopsy There are a variety of ways to interpret a liver biopsy, and the present invention will based on the selected reference level empirically obtained enable the skilled addressee to differentiate between these various stages - among those the most common scoring methods include Metavir and histologic activity index (HAI) also called the Knodell. Alterantives include the Ishak score.
  • HAI histologic activity index
  • Alterantives include the Ishak score.
  • the Metavir scoring system was specially designed for patients with hepatitis C.
  • the scoring consists of using a grading and a staging system.
  • the grade gives an indication of the activity or amount of inflammation and the stage represents the amount of fibrosis or scarring.
  • the grade is assigned a number based on the degree of inflammation, which is usually scored from 0-4 with 0 being
  • HAI histologic activity index
  • the fourth component indicates the amount of scarring in the liver and is scored from 0 (no scarring) to 4 (extensive scarring or cirrhosis). Information about the grade and stage of liver disease is helpful for the healthcare provider and the patient in guiding medical management. For example, treatment is usually indicated if the Metavir score is greater than or equal to 2 or the
  • Ishak/Knodell score is greater than or equal to 3. It is important to know this information in order to help guide management and treatment of HCV. For example, if a medical provider is able to estimate the approximate time when someone became infected with HCV, the results of the biopsy will give an indication of the rate of disease progression : For people with moderate to severe liver damage it is generally recommended that a more aggressive treatment approach is warranted. For people with a milder form of liver disease a watch and wait approach is usually recommended.
  • IP-10 measured e.g. as described in this invention can be used as a non invasive method for treatment guidance.
  • the present invention demonstrates that the reference- level and/or degree of liver fibrosis can be correlated to at least one histological classification selected from the group consisting of METAVIR, Knodell and Ishak. And, in one embodiment, the present invention demonstrates that the reference- level and/or degree of liver fibrosis can be correlated to at least one clinical parameter.
  • Spotting means the application of a blood sample or any other biological fluid or extract to a piece of standardised paper suitable for accurate blood sampling.
  • the sample can be spotted to the filter paper in many ways; the optimal method depends on the clinical setting and desired accuracy.
  • the spotting is done by applying a fixed volume of blood to a piece of paper or by applying blood to the paper until a defined area is covered with the blood. In its most simple embodiment of the present invention, a sample of blood is obtained and a drop of blood is applied to the filter paper.
  • the paper is allowed to dry completely or at least until the sample is essentially dry.
  • Proteolytic activity is partly depending on water content in the sample i.e. high water content in the sample can potentially degrade the IP-10 protein.
  • Essentially dry implies that the sample has a water content which is sufficiently low to prevent sample degradation in the period from sample application on the filter paper until analysis. Wherefore a low water content is prefered for longer storage and better sample stability, but in the case of short time from application on filter paper to analysis, then the requirements for low water content are less strict. Low water content is obtained with drying.
  • the terms 'drying', 'dry', 'essentially dry' and 'completely dry' depends on the relative humidity at site of drying.
  • Relative humidity is a term used to describe the amount of water vapor in a mixture of air and water vapor. It is defined as the ratio of the partial pressure of water vapor in the air-water mixture to the saturated vapor pressure of water at those conditions. The relative humidity of air depends not only on temperature but also on pressure of the system of interest. Relative humidity is normally expressed as a percentage and is calculated as the ratio of the partial pressure of water vapor (H 2 0) in the mixture to the saturated vapor pressure of water at a prescribed temperature.
  • the liquid part of a blood or plasma sample is mainly water, wherefore the filter paper sample can not have a water content below the relative humidity of the local environment after the drying process have been terminated.
  • the drying step may be performed on the lab bench, in a holder, in a rack, in a heating oven, in a sealed plastic bag with dessicator, in a ventilated area (e.g. ; heating oven or a flow bench). Drying can also be done in two steps e.g. first on a lab bench, followed by a second step in a sealed plastic bag with a dessicator. This two step procedure allows for an initial rapid evaporation of large quantities of water flowed by the second controlled drying process to a very low relative humidity (driven by the dessication capability of the dessicator material).
  • Drying can be accelerated by adjusting heat, air pressure, ventialation and other parameters.
  • One of many means to reduce drying time is placing the filter paper in a holder or rack allowing for air circulation from both above and below the spottet sample on filter paper. Under normal laboratory conditions complete drying is achieved within 2-4 hours. But much faster drying times can be obtained by adjusting the parameters listed above. Simple fast drying is possible e.g. in a heated oven with ventilation.
  • the term 'completely dry' does not nessesarily imply that the sample has to have the excact relative humidity as the surrounding atmosphere.
  • the dryness of the sample can be expressed in many ways e.g. relative to the 'relative humidity' or as 'relative humidity' (e.g. % as described above) but also to the 'absolute humidity' (i.e. g (H 2 0)/m 3 (air).
  • the sample is considered essentaily or completely dry if the sample has up to 2 fold higher relative humidity than in the environment where to sample was subjected to drying.
  • the relative humidity of the sample is ⁇ 1.5 fold the relative humidity of the local environment where the drying took place.
  • sample dryness is the absolute humidity.
  • the sample is considered essentaily or completely dry if the sample has an absolute humidity below 5 g/m 3 , such as below 3 g/m 3 or in the range of 5 to 1 g/m 3 .
  • the absolute humidity of the sample is below 1 g/m 3 .
  • Dried samples are either stored immediately at low relative humidity conditions such as but not limited to ⁇ 6% or transported to a storage place for subsequent analysis.
  • the optimal storage is at humid temperature ⁇ +20°C, even more preferred is -20°C or even -70°C.
  • the combination of low humidity and low temperatures is preferred.
  • the humidity can be estimated using various methods the simplest being cards coated with humidity sensitive material - e.g. humidity indicator cards. Low humidity can be assured using desiccator material e.g. silica gel or clay desiccant.
  • desiccator material e.g. silica gel or clay desiccant.
  • the blood, plasma, other biological fluid or extract is dried on a standardized filter paper.
  • a filter paper according to the present invention relates to any semi-permeable paper such as but not limited to Whatman 903, Whatman 3MM and PKU paper used for infant screening.
  • filter papers are made in a varieties of ways since specific applications require specific types of papers.
  • the raw materials might be acid washed wooden fibers, carbon or quartz fibers. Finishing and confectioning is the bulk of the production work.
  • filter paper is usually used with a filter funnel, Hirsch, or Buchner funnel.
  • Ashless filter paper is mainly used for gravimetric methods in quantitative chemical analysis. It has a base weight of 80 g/m2. These papers may be impregnated with various reagents.
  • cellulose based paper or synthetic porous materials or glass based porous material is used.
  • Cotton, Q-Tips and other means that is a semi-permeable barrier placed perpendicular to a liquid or air flow can also be used.
  • Drying the sample can also be done on a non-binding surface e.g. in an eppendoph tube. By drying a drop of sample on a non-binding surface the volume of sample is greater compared to drying on filter paper. Low signals are better detectable using the drying on a non-binging surface variation of the method. When drying on a non-binding surface, the optimal sample is plasma.
  • the filter paper is dried until it is completely dry, preferably at room temperature e.g. 2 to 6 hours. Faster drying time is possible with higher temperature or lower humidity and any means for obtaining such a drying is an object of the present invention.
  • a presently preferred embodiment is drying the filter paper at a low humidity.
  • the filter paper samples are stored in low gas- permeability plastic bags with desiccant added to reduce humidity. Placing the filter paper in a sealed plastic bag with a known volume of desiccator material is a simple way to standardize the drying process. The optimal temperature is minus 80°C, but even prolonged storage at +5°C, ambient temperature and even in tropical climates is possible.
  • the holes in the filter paper are punched with a hole punch such as but not limited to hole puncher, paper puncher, holing pincer, perforator, or any tool that can be used to create holes in sheets of paper.
  • a hole punch such as but not limited to hole puncher, paper puncher, holing pincer, perforator, or any tool that can be used to create holes in sheets of paper.
  • a typical hand held hole punch whether a single or multiple hole punch, has a long lever which is used to push a bladed cylinder straight through the paper. As the vertical travel distance of the cylinder is only a few millimetres, it can be positioned within a centimetre of the lever fulcrum.
  • Another mechanism uses hollowed drills that are lowered by a screwing action into the paper. The paper is cut and forced up into the shaft of the drill to be later used for measurement. This method allows a small machine to cut industrial volumes of paper with little effort.
  • the hole punching is automated and the optimal placement of the hole puncher on the
  • the lab technician separate a small disc of saturated paper from the sheet using an automated or manual hole punch, dropping the disc or discs into a flat bottomed micro titre plate.
  • the hole punching is automated e.g. using the Wallac auto puncher (PerkinElmer, USA).
  • IP-10 the Wallac auto puncher
  • IP-10 also known as C-X-C motif chemokine 10 (CXCL10) or small-inducible cytokine BIO is a protein that in humans is encoded by the CXCL10 gene.
  • the gene for IP-10 is located on human chromosome 4 in a cluster among several other CXC chemokines.
  • IP-10 is secreted by several cell types in response to IFN- Y, TNF-a and other cytokines. These cell types include monocytes, endothelial cells, hepatocytes and fibroblasts. IP-10 elicits its effects by binding to the cell surface chemokine receptor CXCR3.
  • the three-dimensional crystal structure of this IP-10 has been determined under 3 different conditions to a resolution of up to 1.92 A.
  • the Protein Data Bank accession codes for the structures of IP-10 are llv9, lo7y, and lo80.
  • the chemokine IP-10 is determined in combination with other chemokines.
  • IP-10 is determined in combination with another biomarker selected from the group comprising MIG, MCP-2, MCP-1, IL-1RA, IL-2, IFN- ⁇ , MIP-la, ⁇ -lb, SIL-2R, IL-10, TNF-a, SuPAR.
  • another biomarker selected from the group comprising MIG, MCP-2, MCP-1, IL-1RA, IL-2, IFN- ⁇ , MIP-la, ⁇ -lb, SIL-2R, IL-10, TNF-a, SuPAR.
  • Agonist and antagonist IP-10 is determined in combination with another biomarker selected from the group comprising MIG, MCP-2, MCP-1, IL-1RA, IL-2, IFN- ⁇ , MIP-la, ⁇ -lb, SIL-2R, IL-10, TNF-a, SuPAR.
  • IP-10 can be cleaved to form an antagonist from. This can be done e.g. by removing at least 1 amino acid(s) in the N-ternimal of the agonist IP-10 to form an antagonist form.
  • IP-10 can be cleaved by Di Peptidyl Peptidase-4 (DPP4/CD26) which removes the two N-terminal amino acids valine and proline (VP) creating the short IP-10 (aa3-77). More extensive N-terminal reduction of IP-10 can also occur, these even shorter forms of IP-10 appear also to have antagonist function.
  • DPP-4 Di Peptidyl Peptidase-4
  • VP valine and proline
  • More extensive N-terminal reduction of IP-10 can also occur, these even shorter forms of IP-10 appear also to have antagonist function.
  • the enzymatic activity of DPP-4 can be blocked by inhibitors such as Stiagliptin. Other protease inhibitors such as the protein inhibitor cocktails from Roche are also usefull.
  • the IP-10 level determined in the sample is shorter forms of the IP-10 molecule such as but not limited N-terminal truncated IP-10 e.g. aa3-77.
  • the IP-10 level determined in the sample is described by the biologic function of the molecule, such as the antagonist form. Other preferred embodiments determine the degree of citrinulated IP-10.
  • the IP-10 level relates to the level of the antagonist form of IP-10 and/or the level of the agonist form of IP-10.
  • the IP-10 measured is the IP- 10 level of either one of the forms of IP-10 or the level of both the agonist and the antagonist (the sum of both forms).
  • the IP-10 level refers to the level of the agonist form of IP-10.
  • An important advantage with the filter paper method compared to all conventional pre-analytical storage/transport of samples is that the drying of the sample stops enzyme activity and inhibits extra corporal truncation and other changes of plasma proteins such as the IP-10 molecule.
  • IP-10 can be measured both as agonist IP-10, antagonist IP-10 and/or both forms of IP-10 at the same time.
  • the proportion of antagonist form of IP-10 to agonist from of IP-10 depends on CD26 activity.
  • IP-10 Advanced HCV infection has higher levels of IP-10, but also higher expression of CD26.
  • Antagonist form is related to different clinical presentations of HCV infection such as but not limited to more chronic HCV infection, advanced fibrosis and poor outcome. Specific cleaved forms of IP-10 are valuable biomarkers.
  • the present invention relates to a method as disclosed herein, where the IP- 10 level relates to the level of the antagonist form of IP-10 and/or the level of the agonist form of IP-10.
  • the antagonist form of IP-10 is specifically determined.
  • the agonist from of IP-10 is determined and in yet a preferred embodiment the ratio of antagonist to agonist IP-10 is used as a monitor of the level of IP-10.
  • the levels of one or more forms of IP-10 are further related to the expression and/or function and/or enzymatic activity of CD26.
  • CD26 activity in the sample can be determined by comparing the two levels of IP-10 antagonist measured with the time frame between.
  • IP-10 level relates to the level of the antagonist form of IP-10 and/or the level of the agonist form of IP-10.
  • the genetic makeup of the infected host is determined from host DNA from the cells in the dried blood spot sample.
  • certain genomic polymorphisms are determined in the blood spotted on filter paper.
  • SNPs related to immunity can be measured in the dried blood spots. Depending on HCV genotypes and target population the SNPs vary.
  • the SNP is selected from the group consisting of IL-28b SNPs rs8099917, rsl2979860 and rsl2980275 genotypes.
  • polymorphism of OAS-1, CTGF, IL-28A and IL-29 are measured.
  • the preferred genotype subjected for SNP analysis depends on the area. E.g. in Japanese patients rs8099917 carries most clinical relevant information, in Caucasians rsl2979860 is the desired SNP target.
  • the allele of rsl2979860 is determined.
  • the TT, CT or CC allele of rsl2979860 the HCV patients determines, which IP-10 reference should be used for measurements.
  • the rsl2979860 allele TT is a predictor of poor treatment response and is linked to progression in disease, CC is linked to increased treatment success.
  • TC is somewhere in between.
  • the determination of the genotype is done using genotyping, e.g. using PCR.
  • PCR can be done from DNA extracted from the filter paper using methods known to the skilled addressee.
  • the filter paper sample used for genotyping is punched from the same spotted sample used for IP-10 determination.
  • the genotype is constant and with serial or subsequent
  • one object of this invention relates to a method, wherein the IP-10 level is optionally combined with a concomitant assessment of IL28b-related SNPs.
  • the filter paper sample used for genotyping is sent to a separate lab for determination.
  • HCV virus is readily detectable from dried blood and dried plasma spots wherefore
  • IP-10 detection can be combined with HCV viral genotyping and viral load monitoring. Acoordingly, in one embodiment of the present invention, the IP-10 level is combined with a concomitant assessment of HCV viral genotype. In another embodiment, wherein the IP-10 level is optionally combined with a concomitant assessment of HCV viral load. In a further embodiment, the IP-10 level is combined with one or more of a concomitant assessment of IL-28b-related SNPs, HCV viral genotype and HCV viral load.
  • IP-10 is a correlate for liver fibrosis also in other diseases such as hepatitis B infection, primary biliary cirrhosis and autoimmune hepatitis. In these and other diseases liver fibrosis can be monitored by determining the IP-10 level using the filter paper technique as described herein.
  • IP-10 may be determined at the level of the molecule itself or to the extent to which a gene is expressed.
  • the level of IP-10 is measured by conventional analytical methods As stated above, detection of IP-10 may be made at the protein or nucleic acid levels. Consequently, reference to presence or level of said immune effector molecule includes direct and indirect data. For example, high levels of IP-10 mRNA are indirect data showing increased levels of IP-10. Antibodies to the immune effecters are particularly useful.
  • IP-10 can be determined at RNA, protein or other levels by any cytokine or chemokine detection method known to the skilled addressee such as but not limited to xMAP, multiplexing, Luminex, ELISA, Chemiluminesence. Other techniques such as molecular methods, e.g. chips, mass spect, qPCR and PCR are available. Most preferably IP-10 is determined with a sandwich mAb technique where one of the mAbs is linked to an enzyme, a chemoluminecent, a fluorescent or other substances with the ability to generate an amplified signal. On the protein level IP-10 can be detected as agonist, antagonist and/or total IP- 10. Agonist level can be determined by measuring total IP-10 subtrated
  • Antagonist level can be determined by measuring total IP-10 subtrated agonist level IP-10.
  • Total IP-10 can be determined by adding the agonist IP-10 level to antagonist IP-10 level. More complex algorithms included determining the DPP4 level in the DPS sample and combining this with the level of one or more forms of IP-10.
  • Elution is the extraction of plasma proteins such as biomarkers from the filter paper.
  • the biomarker in the sample is eluted in a buffer.
  • This buffer could be water, saline, pH buffered saline, or saline added a detergent (e.g. tween 80, tween 20), sodium azide, bronidox and/or bovine serum albumin.
  • elution is done with a protease inhibitor or protease inhibitor cocktail added to the buffer.
  • the protease inhibitor is specific for di-peptidyl peptidase 4 aka DPP-4 aka CD26.
  • the protease inhibitor is Sitagliptin. Elution can be improved with shaking, (e.g.
  • the biomarker is eluted and detected in one step. This is achieved by performing the elution in a well coated with mAbs and the buffer containing a mAb coupled with an enzyme or other substance that allows amplification of the signal.
  • the elution is done directly in an ELISA plate coated with monoclonal antibodies specific for the biomarker one wishes to determine.
  • the elution is done in a buffer comprising the detection antibody coupled to an enzyme.
  • the elution is done in a coated ELISA plate, after washing the detection mAb is then added.
  • elution is done in a buffer comprising beads that substitute the solid phase (e.g. the plastic surface of the ELISA plate) this method could be
  • a reference level is a level used to compare a measurement of IP-10 to clinical meaningful information.
  • a reference level can represent the state of HCV fibrosis. Reference levels are defined before the measurement of the sample and can both be from the same patient, from a representative cohort of patients with known fibrosis stage (e.g. defined using pathology (biopsy and histology), imaging, endoscopy or transient elastography) or from patients representing a known subpopulation with high risk of progression in HCV fibrosis. In a preferred example a subpopulation comprise patients with certain genomic polymorphisms such as IL-28b SNPs.
  • One such reference level can be set by comparison of the IP-10 level to
  • liver fibrosis is scored e.g. using the
  • METAVIR score This scoring system assigns two standardized numbers: one to represent the degree of inflammation and the other the degree of fibrosis.
  • the fibrosis is graded on a 5-point scale from 0 to 4.
  • the activity which is the amount of inflammation (specifically, the intensity of necro-inflammatory lesions), is graded on a 4-point scale from AO to A3.
  • Transient elastography is a rapid, non-invasive, bedside method to evaluate liver fibrosis by measuring liver stiffness. This device is based on one- dimensional transient elastography, a technique that uses both ultrasound (5 MHz) and low-frequency (50 Hz) elastic waves, whose propagation velocity is directly related to elasticity.
  • fibrosis for Fibroscan examples include METAVIR score F0/F1: ⁇ 7.1 kPa, F1/F2 7.1-8.7kPa, F2 8.7-9.5 kPa, F3 9.5- 12.5 kPa, F3/F4 12.5-14.5 kPa, Cirrhosis 12.5 to 75.5 kPa.
  • F0/F1 ⁇ 7.1 kPa
  • F1/F2 7.1-8.7kPa F2 8.7-9.5 kPa
  • F3/F4 12.5-14.5 kPa Cirrhosis 12.5 to 75.5 kPa.
  • Cirrhosis 12.5 to 75.5 kPa.
  • blood biomarkers such as but not limited to plasma proteins, aminotransferases, alkaline phosphatase, bilirubin, albumin, prothrombin time, globulins, serum sodium, thrombocyte count, leukocyte count and neutrophilocyte count, coagulation factors, serological markers for hepatitis viruses,
  • autoantibodies anti-smooth muscle, anti-mitochondria, anti-LKM
  • ferritin and transferrin saturation markers of iron overload
  • copper and ceruloplasmin markers of copper overload
  • immunoglobulin levels IgG, IgM, IgA
  • cholesterol glucose and alpha 1-antitrypsin
  • the reference level is defined using a combination of biomarkers e.g. FibroTest, actitest, fibrospect, HCV-fibroSure.
  • the reference- 1 eve I is obtained from a spotted blood sample previously obtained from said patient.
  • the reference- level is obtained from a cohort of patients with hepatitis C induced liver fibrosis.
  • the reference- 1 eve I and/or degree of liver fibrosis is correlated to at least one histological
  • METAVIR Knodell and Ishak
  • fibroscan liver stiffness classification selected from the group consisting of METAVIR, Knodell and Ishak, or a fibroscan liver stiffness classification.
  • a simple way to set a reference level would be to determine the level of IP-10 using the method disclosed in a representative cohort of patients with known METAVIR scores F0-F4, liver stiffness measurements or clinical endpoints such liver cancer and death.
  • a representative range of IP-10 concentrations can be determined as correlate for e.g. a METVIR score.
  • a reference for F0/F1 would be ⁇ 30pg/ml and F4 would be >71pg/ml.
  • Other assays will render other levels of IP-10 from the same samples wherefore these reference values can change.
  • One embodiment of the invention relates to a reference level wherein the reference- 1 eve I is obtained from a spotted blood previously obtained from said patient.
  • the progression in liver fibrosis of a patient is determined by comparing a previous determined level of IP-10 (reference level) to a measurement in the same patient.
  • the timeframe between a first and a second sample preparation is 1 week to years.
  • the reference level could comprise an average of at least 2 previous determined IP-10 levels in the patient.
  • the time between reference sample and sample is 14 days to 2 years.
  • the kinetics of the development in liver fibrosis can be evaluated.
  • IP-10 An increase in IP-10 from the reference will indicate fibrosis progression.
  • IP-10 agonist and antagonist forms are determined and used in combination with total IP-10 levels.
  • other biomarkers such as genotype (e.g. IL-28b SNPs), HCV genotype and plasma proteins are combined with the determined IP-10 level to further improve diagnostic accuracy or disease monitoring.
  • the reference level is dependent on co-morbidity e.g. HIV infection, HBV infection and other; age, sex and other determinants.
  • the term "reference” relates to a standard in relation to quantity, quality or type, against which other values or
  • characteristics can be compared, such as e.g. a standard curve or a METAVIR score.
  • One embodiment of the invention relates to a reference level wherein the reference- 1 eve I is obtained from a spotted blood previously obtained from said patient.
  • the reference- 1 eve I is obtained from a cohort of patients with hepatitis C induced liver fibrosis.
  • One embodiment of the present invention contemplates a method comprising collecting a sample from a subject wherein said sample comprises immune effecter-molecules and then measuring the presence of or elevation/decrease in the level of an immune effecter molecule, especially IP-10.
  • the sample is derived from the group consisting of blood, serum, plasma, urine, pleural fluid, bronchial fluid, oral washings, tissue biopsies, ascites, pus, cerebrospinal fluid, aspitate, liver biopsy material and follicular fluid.
  • the sample is derived from blood.
  • the sample may however also comprise cells selected from the group consisting of whole blood, mononuclear cells from pleural fluid, peripheral blood
  • PBMC mononuclear cells
  • T cells CD4 T cells
  • CD8 T cells gamma-delta T cells
  • monocytes macrophages
  • granolocytes granolocytes
  • NK cells NK cells.
  • the sample can also comprise hepatocytes e.g. homogenized hepatocytes.
  • hepatocytes e.g. homogenized hepatocytes.
  • the present invention extends to other samples containing immune cells such as but not limited to pleural fluid, ascites fluid, lymph fluid, spinal or cerebral fluid, tissue fluid and respiratory fluid including nasal, and pulmonary fluid.
  • Aliquots of blood may be in volumes ranging from 10 ⁇ _-500 ⁇ , such as but not limited to 5 ⁇ _, 10 ⁇ _, 15 ⁇ _, 25 ⁇ _, 30 ⁇ _, 35 ⁇ _, 40 ⁇ _, 50 ⁇ _, 60 ⁇ _, 75 ⁇ _, 80 ⁇ _, 100 ⁇ , 200 ⁇ , 300 ⁇ , 400 ⁇ , 500 ⁇ . Volumes can also be determined as drops of blood or determined by covering a specified area of the filter paper corresponding to a volume.
  • the sensitivity of any given test define the proportion of individuals with a positive response who are correctly identified by the test, e.g. the sensitivity is 100%, if all individuals with a given condition such as liver cirrhosis have a positive test.
  • the specificity of a given test reflects the proportion of individuals without the condition who are correctly identified by the test, e.g. 100 % specificity is, if all individuals without the condition e.g. liver fibrosis have a negative test result.
  • Sensitivity is defined as the proportion of individuals with a given degree of liver fibrosis, who are correctly identified by the described methods of the invention (e.g. has a IP-10 level in a present range which corresponds to the liver fibrosis stage e.g. defined by liver biopsy).
  • Specificity herein is defined as the proportion of individuals without a given degree of liver fibrosis, who are correctly identified by the described methods of the invention (e.g. has a IP-10 measurement outside of the range of the liver fibrosis stage).
  • ROC receiver-operating characteristics
  • the clinical performance of a laboratory test depends on its diagnostic accuracy, or the ability to correctly classify subjects into clinically relevant subgroups.
  • Diagnostic accuracy measures the test's ability to correctly distinguish two different conditions of the subjects investigated. Such conditions are for example no fibrosis, mild fibrosis and advanced fibrosis.
  • the ROC plot depicts the overlap between the two distributions by plotting the sensitivity versus 1 - specificity for the complete range of decision thresholds. On the y-axis is sensitivity, or the true-positive fraction [defined as (number of true-positive test results) (number of true-positive + number of false- negative test results]. This has also been referred to as positivity in the presence of a disease or condition. It is calculated solely from the affected subgroup.
  • the ROC plot is independent of the prevalence of disease in the sample.
  • Each point on the ROC plot represents a sensitivity/- specificity pair corresponding to a particular decision threshold.
  • a test with perfect discrimination has an ROC plot that passes through the upper left corner, where the true- positive fraction is 1.0, or 100% (perfect sensitivity), and the false- positive fraction is 0 (perfect specificity).
  • the theoretical plot for a test with no discrimination is a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall in between these two extremes.
  • Clinical utility of the marker may be assessed in comparison to and in combination with other diagnostic tools for the given condition.
  • a method comprising constructing a percentile plot of the IP-10 level obtained from e.g. a healthy population ad constructing a ROC (receiver operating characteristics) curve based on the IP-10 level determined in the healthy population and on the IP-10 level determined in HCV patients with a given degree of fibrosis, selecting a desired specificity and determining from the ROC curve the sensitivity corresponding to the desired specificity, determining from the percentile plot the IP-10 level corresponding to the determined sensitivity; and predicting the individual.
  • the level of IP-10 is compared to a set of reference data or a reference value such as the cut-off value to determine whether the subject is at an increased risk or likelihood.
  • IP-10 may be compared to a set of reference data to determine whether the subject is likely to advance in the extend of fibrosis or remain stable
  • This cut-off must be established by the laboratory, the physician or on a case by case basis by each patient.
  • cut point can be determined as the mean, median or geometric mean of a HCV uninfected negative control group (or any group representing a given METAVIR score) +/- one or more standard deviations or a value derived from the standard deviation.
  • IP-10 measurements are combined with measurements of one or more other biomarkers, and compared to a combined reference-level.
  • the measured biomarker levels can be combined by arithmetic operations such as addition, subtraction, multiplication and arithmetic
  • Cut-off points can vary based on specific conditions of the individual tested such as but not limited to the risk of having the disease, occupation, geographic residence or exposure.
  • Cut-off points can vary based on specific conditions of the individual tested such as but not limited to age, sex, genetic background (i.e. HLA-type), acquired or inherited compromised immune function (e.g. HIV infection, diabetes, patients with renal or liver failure, patients on treatment with immune-modifying drugs such as ribavirin or peg-intron).
  • HLA-type genetic background
  • acquired or inherited compromised immune function e.g. HIV infection, diabetes, patients with renal or liver failure, patients on treatment with immune-modifying drugs such as ribavirin or peg-intron.
  • methods of the present invention are processes of decision making by comparison.
  • reference-values based on subjects having the disease or condition of interest and/or subjects not having the disease, infection, or condition of interest are needed.
  • the cut-off level (or the cut-off point) can be based on several criteria including the number of subjects who would go on for further invasive diagnostic testing, the average risk of having and/or developing e.g. cirrhosis to all the subjects who go on for further diagnostic testing, a decision that any subject whose patient specific risk is greater than a certain risk level such as e.g. 1 in 400 or 1 : 250 (as defined by the screening organization or the individual subject) should go on for further invasive diagnostic testing (e.g. biopsy) or other criteria known to those skilled in the art.
  • a certain risk level such as e.g. 1 in 400 or 1 : 250 (as defined by the screening organization or the individual subject) should go on for further invasive diagnostic testing (e.g. biopsy) or other criteria known to those skilled in the art.
  • the cut-off level(s) can be adjusted based on several criteria such as but not restricted to certain group of individuals tested. E.g. the cut-off level could be set lower in patients at great risk of accelerated progression in fibrosis, cut-offs may be higher in groups of patients with low risk of progression in fibrosis.
  • the cut off level(s) can also be adjusted based on the viral genotype, viral load, liver enzyme levels (e.g. ASAT / ALAT), host IL-28b genotype. In other embodiments the cut off level(s) depend on the agnotist IP-10 level and/or the antagonist IP-10 levels and/or the agonist/antagonist ratio or derived values.
  • the discriminating value is a value which has been determined by measuring the parameter or parameters in both a healthy control population and a population with known fibrosis thereby determining the discriminating value which identifies the infected population with either a predetermined specificity or a predetermined sensitivity based on an analysis of the relation between the parameter values and the known clinical data of the healthy control population and the fibrosis patient population, such as it is apparent from the detailed discussion in the examples herein.
  • the discriminating value determined in this manner is valid for the same experimental setup in future individual tests.
  • the level threshold of IP-10 useful as cut-off value(s) for liver fibrosis are: no or minimal fibrosis (METAVIR F0/F1) 0-30pg/ml, some fibrosis (METAVIR F2) 31-50pg/ml, bridgeing fibrosis (METAVIR F3) 51-70pg/ml and advanced fibrosis or cirrhosis (METAVIR F4) >71pg/ml.
  • a single cut off level e.g. at 30pg/ml can be a guidance tool for more frequent check-ups. Similar e.g. 50pg/ml cut off could be used to indicate when a patient should be offered treatment.
  • cut off is in the range of 10-200pg/ml.
  • cut offs set for plasma samples can be converted to filter paper samples simpy by comparing the volume of plasma used for the analysis with estimates of the volume available in the filter paper used for analysis.
  • the signal intensity from 2 filter paper discs corresponds to a volume of blood or plasma of
  • 47.6mm 2 i.e. 1 ⁇ _ plasma generates a signal corresponding to 13.2mm 2 of dried plasma or blood or dried blood spot if analysed in a 100 ⁇ _ ELISA well.
  • IP-10 is determined in shorter forms such as but not limited to aa3-77 IP-10, then other cut offs apply. In general cut offs for total IP-10 are higher than cut offs determined with subpopulations of the total amount of IP-10 protein.
  • Calculating the slope of this curve using regression analysis can help in the assessment of the progression in fibrosis.
  • IP-10 of lOpg/ml over a 6 months period could result in further examinations such as liver biopsy.
  • a constant 5pg/ml increase in IP-10 levels in 8 consecutive visits over a 10 year period can guide the assessment of when the patient should be signed up for liver transplant or anti-viral treatment.
  • Serial measurements by the patient e.g. on a daily or weekly basis can be used to monitor e.g. the first weeks of anti-viral treatment. This method is particularly good for assessing treatment effect and predicting the probability of success.
  • the multivariate DISCRIMINANT analysis and other risk assessments can be performed on the commercially available computer program statistical package Statistical Analysis System (manufactured and sold by SAS Institute Inc.) or by other methods of multivariate statistical analysis or other statistical software packages or screening software known to those skilled in the art.
  • the present invention relates to the correlation of IP-10 with the degree of liver fibrosis, thus in one embodiment the method demonstrates that the reference- 1 eve I and/or degree of liver fibrosis is correlated to at least one histological classification selected from the group consisting of METAVIR, Knodell and Ishak. Time frame
  • the invention relates to method wherein the timeframe between a first and a second sample preparation is 1 week to years.
  • filter paper for spotting blood for subsequent analysis is well known, e.g. for analysis of blood samples of new born babies for inherited metabolic diseases.
  • the advantages of this are good preservation of blood constituents, easy transport and facile long term storage.
  • the filter papers of the present invention may be used immediately or after a short period of time such as after 10 min. for analysis or stored for subsequent analysis for days, weeks and years. In particular the filter paper may be transported by ordinary mail over a distance before storage or analysis in a laboratory.
  • the filter papers of the present invention can be stored between 10 min and up to 50 years.
  • Hepatitis C genotype 1 hepatitis C RNA PCR- detectable for more than 6 months
  • Group 1 had no/mild fibrosis
  • group 2 had cirrhosis.
  • Fibrosis/chirrosis was determined by a successful examination with Transient Elastoraphy using FibroScan® (Echosens, Paris, France).
  • blood was drawn for hepatitis C RNA detection, platelet count, analysis of alanine aminotransferase (ALT) and a plasma sample was frozen for the later analysis of IP-10.
  • ALT alanine aminotransferase
  • Filter paper For these experiment we used various types of filter paper: Whatman 903 (Whatman, USA), Whatman 3MM (Whatman, USA), normal toilet paper (Irma, Copenhagen), unbleached news paper paper (Politiken, Copenhagen) and the filter paper used in the national PKU test supplied from SSI Copenhagen (SSI, Denmark).
  • Hole punch Discs were cut from the filter paper using a normal (IS0838) stationary 6.0mm hole punch (Impega, Bog og Ide, Copenhagen, Denmark), a pair of scissors (Fiskars, Finland) or a 3mm single hole punch (Millipore, USA).
  • Biomarker determination The optimal method for generating strong, reproducible and faster results in the filter paper method was the optimized direct elution and incubation method. In this method the discs were placed directly in the coated ELISA wells in a buffer containing the secondary-HRP conjungated mAb (detection mAb).
  • the biomarker was extracted from the discs in the buffer for 1 to 24 hours where after the buffer was transferred without the discs to the ELISA plate and incubated as described, this approach took longer time and was more laborious. Incubation was done for 2 hours, but in some experiments from 30 min to 6 hours.
  • the preferred buffer was a PBS buffer with 2% bovine serum albumin and 1% Tween-20. Following 1 to 4 washes with PBS 0.1%
  • Example 1 For the determination of IP-10 levels in plasma, the sample was diluted 1 :4 in the 2% BSA, pH 7.4 PBS buffer and analysed in duplicates according to the steps described in the above mentioned protocol.
  • Example 1 For the determination of IP-10 levels in plasma, the sample was diluted 1 :4 in the 2% BSA, pH 7.4 PBS buffer and analysed in duplicates according to the steps described in the above mentioned protocol.
  • IP-10 is a fibrosis marker that can be measured in plasma.
  • Figure 1 compares the two groups of patients with HCV; one with little/no fibrosis and one with advanced liver fibrosis. As shown there is a difference between the two groups of patients.
  • IP-10 is a fibrosis marker that can be measured after drying on filter paper.
  • Figure 2 compares the two groups of patients with HCV; one with little/no fibrosis and one with advanced liver fibrosis. As shown there is a difference between the two groups of patients.
  • Example 4
  • the fibrosis stage specific discriminatory potential is high and comparable with plasma and filter paper method.
  • Figure 4 is a receiver operation characteristic curve analysis comparing the ability of the plasma and the filter paper method of IP-10 determination to differentiate between little/no fibrosis (specificity) and cirrhosis (sensitivity). As can be appreciated from the figure both methods are able to differentiate between the two groups of patients, if further appears that the filter paper method is superior to the plasma method, although this difference failed to reach significance in this material (Hanley-McNeal's test).
  • IP-10 can be detected in both spotted plasma and spotted blood
  • Table 1 lists 6 samples A-F.
  • A-B sample was a normal heparin sample from healthy donors, C-F samples are whole blood stimulated with CMV peptides and LPS. Each blood sample was divided in two parts. From the first part 2 spots of
  • Results table 2-5 This un-optimized system enables stabile storage oF DPS samples for at least 4 weeks at (at least) 37°C. Similar results were obtained for whole blood (data not shown).

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Abstract

La présente invention porte sur un procédé pour surveiller le degré de fibrose hépatique chez un patient infecté par le virus de l'hépatite C par observation d'un échantillon de sang obtenu à partir d'un humain infecté par le virus de l'hépatite C sur un papier filtre pour une analyse ultérieure de la corrélation entre le niveau IP-10 et le degré de fibrose hépatique.
PCT/DK2012/050127 2011-04-13 2012-04-13 Surveillance de la fibrose hépatique chez un patient infecté par le virus de l'hépatite c WO2012139591A1 (fr)

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WO2008064684A1 (fr) 2006-12-01 2008-06-05 Statens Serum Institut Procédé de criblage utilisant l'adsorption d'échantillon sur un papier filtre
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